CN1233291A - Method for recombinant adeno-associated virus-directed gene therapy - Google Patents

Abstract

A method of prolonging gene expression by reducing immune response to a recombinant adeno-associated virus (AAV) bearing a desired gene administered into the muscle of a mammal is described.

Description

The method of recombinant adeno-associated virus-directed gene therapy

This work obtains the support of the DK47757 fund that national institute of health authorizes.United States Government enjoys part right of the present invention.

Background of invention

Adeno associated virus (AAV) is the replication defect type parvovirus, and its genome is about 4.6kb, comprises the reverse terminal repeat (ITR) of 145 Nucleotide.The single stranded DNA genome of AAV contains the gene (rep and cap) that duplicates and form virion.

Behind this avirulence Human virus infected person cell, viral genome is integrated in the karyomit(e) 19, causes the cell latent infection, does not produce the virus of infection, does not also duplicate, and removes acellular cleaved property helper virus (as adenovirus or simplexvirus) coinfection simultaneously.After being infected by helper virus, the AAV provirus obtains saving and being amplified, thereby has produced AAV and helper virus.

AAV has unique feature, and promptly it can be used as attractive carrier and is used for foreign DNA is delivered in the cell.Many experiments group after deliberation the potential application of AAV in the treatment disease.

The in vitro study of reorganization AAV (rAAV) is disappointed, because its transduction frequency is very low; In the presence of wild-type AAV that does not have contaminative or auxiliary adenovirus, make cell cultivate recombinant gene expression [D.Russell etc., Proc.Natl.Acad.Sci.USA, the 91:8915-8919 (1994) that only follows seldom with rAAV; I.Alexander etc., J.Virol., 68:8282-8287 (1994); D.Russell etc., Proc.Natl.Acad.Sci.USA, 92:5719-5723 (1995); K.Fisher etc., J.Virol., 70:520-532 (1996); With F.Ferrari etc., J.Virol., 70:3227-3234 (1996)].And when rep did not exist, the effect of integration was very low, and can not be positioned karyomit(e) 19[S.Kumar etc., J.Mol.Biol., 222:45-57 (1991)].When adenovirus existed, the transduction of AAV significantly strengthened, because strand rAAV genome is transformed into nonconformable, double-stranded intermediate product [K.Fisher etc., J.Virol., the 70:520-532 (1996) with transcriptional activity; With F.Ferrari etc., J.Virol., 70:3227-3234 (1996)].Adenovirus has strengthened transduction [K.Fisher etc., J.Virol., the 70:520-532 (1996) of rAAV by the expression of early gene product E4 ORF6; With F.Ferrari etc., J.Virol., 70:3227-3234 (1996)].

Now in gene therapy body inner model, sneak into rAAV and played function vector.Most promising result is in central nervous system, has realized stable transduction [M.Kaplitt etc., Nat.Genet., 8:148-154 (1994)] in the postmeiotic cell at this place.Medullary cell and rAAV in vitro cultivate meeting and produce some transductions, and effective hematopoiesis moves into [J.Miller etc., Proc.Natl.Acad.Sci.USA, 91:10183-10187 (1994) although there is not proof to have stable in transplantation model; G.Podsakoff etc., J.Virol.68:5656-5666 (1994); With C.Walsh etc., J.Clin.Invest., 94:1440-1448 (1994)].

Giving rAAV to air flue or blood can make gene change pulmonary epithelial cells [K.Fisher etc., J.Virol., 70:520-532 (1996) respectively over to; With T.Flotte etc., Proc.Natl.Acad.Sci.USA, 90:10613-10617 (1993)] and liver cell [K.Fisher etc., J.Virol., 70:520-532 (1996)] in; Yet, find that transgene expression level is very low, unless there is adenovirus to have [K.Fisher etc., J.Virol., 70:520-532 (1996)].

Required is the transgenic method that improves the rAAV-mediation.

Summary of the invention

The invention provides the method that the selected gene of a kind of improvement (this gene passes to animal by reorganization AAV) is expressed.Method is included in does not have helper virus to exist down, will comprise required genetically modified reorganization AAV carrier and import among the myocyte.This carrier can give cardiac muscle, unstriated muscle or skeletal muscle preferably.

In a preferable example, a kind of useful secreting and/or diffusible product in treatment of the transgenes encoding that rAAV transmits.In this example, transgene product has result of treatment to the position of distance site of delivery certain distance.In another example, transgenes encoding need pass to muscle (for example be used for treating muscular dystrophy) can not excretory product (as the dystrophin polypeptide).

The present invention provides a kind of treatment animal haemophiliachemophiliac method on the other hand.Method comprises the adeno associated virus that animal muscle is recombinated, and this virus comprises the gene of coding factor IX and the sequence of regulating this genetic expression.

A further aspect of the invention provides the treatment animal atherosclerotic method.This method comprises the adeno associated virus that gives the animal muscle reorganization, and this virus comprises the gene of the ApoE that encodes and the regulating and controlling sequence of energy expressing said gene.

In the detailed description of preferred embodiments of the present invention, will further specify others of the present invention and advantage.

The accompanying drawing summary

Fig. 1 shows the line style arrangement architecture of AAV.CMVLacZ (4883bp).Related elements comprises AAVITR (solid black surround), CMV promotor (being with hatched arrow), SV40 intron and polyadenylation signal (hollow frame) and lacZ DNA (band point square frame).Also show the position of the cDNA probe that is used for detecting inner BamH I fragment and total length carrier in addition.

Fig. 2 A shows the line style of AAV.CNVLacZ concatermer (concatomer) and arranges.Relevant mark comprises AAV ITR (being with hatched square frame), cmv enhancer/promotor (filled black arrows), SV40 intron and polyadenylation signal (hollow square frame) and lacZ cDNA (band point square frame).AAV.CMVLacZ monomer shown in the figure is that (mark j place) connects by the mode of direct end to end at the ITR place.Therefore, in sketch, the AAV ITR of two copies is arranged in the joint.

Fig. 2 B shows the enlarged view of connecting zone.The explanation of mark of correlation is in above-mentioned Fig. 3 A.Horizontal arrow is represented to be used for to increase across the position and the direction of the PCR primer of provirus joint.Primer 005 is the sense strand primer.Primer 013 and 017 is the antisense strand primer.

Fig. 3 shows and adopts the PCR product of estimating behind the directly terminal monomer A AV.CMVLacZ genome that end is connected in series.Joint (mark J place) shows two complete ITR (hatched square frame is arranged) and separately " FLOP " and " FLIP " orientation thereof.Also show CMV promotor (solid black surround) and polyadenylation signal (hollow square frame) in addition.PCR clone side joint EcoR I site, position (as shown in the figure) in the pCR II.Also show three SnaB I sites that are arranged in the PCR product among the figure.Also show primer 005 and 013.

Fig. 4 A has described the structure of PCR product with Fig. 4 B-4G, this structure be shown in Fig. 4 A the AAV.CMVLacZ concatermer of head-to-tail.Fig. 4 A has shown the genomic expectation of the monomer A AV.CMVLacZ PCR product that adopts direct end that end is connected in series.Joint (mark j place) shows two complete ITR (hypographous square frame) and separately " FLOP " and " FLIP " orientation thereof.Also show CMV promotor (solid black surround) and polyadenylation signal (hollow square frame) in addition.

Fig. 4 B represents to clone the structure of 3 PCR product.

Fig. 4 C represents to clone the structure of 8 PCR product.Clone 8 size is almost equal with clone 3, but different ITR joint arrangement modes is arranged.

Fig. 4 D represents to clone the structure of 5 PCR product.

Fig. 4 E represents to clone the structure of 2 PCR product.

Fig. 4 F represents to clone the structure of 6 PCR product.

Fig. 4 G represents to clone the structure of 7 PCR product.

Fig. 5 A represents the activation at the cytotoxic T lymphocyte of adenovirus antigen and lacZ.This is the analysis that the lymphocyte from 5 group of 1 results of embodiment is carried out.

Fig. 5 B represents the activation at the cytotoxic T lymphocyte of adenovirus antigen and lacZ.This is the analysis that the lymphocyte from 5 group of 2 results of embodiment is carried out.

Fig. 5 C represents the activation at the cytotoxic T lymphocyte of adenovirus antigen and lacZ.This is the analysis that the lymphocyte from 5 group of 3 results of embodiment is carried out.

Fig. 6 A represents among 5 groups of 1-4 of embodiment to comprising the not lymphocytic activation situation of T in the synantigen responsing reaction of beta-galactosidase enzymes, purifying AAV or adenovirus.The secretion of IFN-γ by representing T cell TH1 subgroup proves this kind activation.

Fig. 6 B represents that 5 groups of 1-4T lymphocytes of embodiment are to the activation situation in the different antigenic responsing reaction that comprises beta-galactosidase enzymes, purifying AAV or adenovirus.The secretion of IL-10 by expression T cell TH2 subgroup proves this kind activation.

Fig. 7 A represents the result of enzyme-linked immunosorbent assay (ELISA), and it has shown the generation of the antibody of anti-beta-galactosidase enzymes in embodiment 5 each group.

Fig. 7 B represents the result of enzyme-linked immunosorbent assay (ELISA), and it has shown the generation of the antibody of anti-5 type adenovirus in embodiment 5 each group.

Fig. 8 is IM injection 2 * 10 in each animal (n=4) ¹¹RAAV-hF. after the IX vector gene group, the synoptic diagram that the hF. IX plasma concentration versus time in the C57BL/6 mouse changes.

Fig. 9 represents to the circulating antibody into the F. IX of resisting that produces after the IM injection rAAV-hF. IX in the C57BL/6 mouse.Use ELISA, as standard substance, be determined in each animal and inject 2 * 10 with the anti-hF. IX of mouse MAb [Boehringer Mannheim] ¹¹Anti-hF. IX antibody concentration changing conditions in time in the blood plasma of vector gene group (n=3) back.Every line is represented an animal.

Figure 10 represents that the hF. IX plasma concentration of three mouse is with the injection changing conditions of back time.Every kind of mark is represented different animals.The 4th animal carries out during 5 weeks after the traumatic bloodletting dead after injection in this experiment.

The hF. IX plasma concentration that Figure 11 represents four Rag-1 mouse with injection rAAV-hF. IX after the changing conditions of time.The different animal of each mark representative.

Figure 12 represents that rAAV head in the transducer cell is to tail concatermer synoptic diagram.

Figure 13 represents the building process of AV.CMVApoE.

Detailed Description Of The Invention

The invention provides the transgenosis method that is oriented to muscle of a kind of adeno-associated virus mediation, the method can not have under helper virus or external source accessory molecule, high level, express transgenic stably. The method is particularly related to and imports the myocyte with carrying required genetically modified restructuring AAV. With the rAAV carrier according to being injected directly into like that in cardiac muscle, skeletal muscle or smooth muscle of hope, transgenosis encode secretion and/or diffusible treatment product (as polypeptide or RNA molecule). Yet, the present invention also can be used for similarly (injection) encode non-secretory, the nucleic acid for the treatment of purposes product is arranged.

Particularly, the purifying rAAV (that is, there is no by the rAAV of adenovirus or wild type AAV pollution) that present inventor's discovery, intramuscular injection do not contain adminicle can cause muscle fibre efficiently to be transduceed, thereby render transgenic is expressed stable and extended. " do not contain adminicle " and also refer in AAV there is no helper virus or other external source accessory molecule (that is, being not natural all or accessory molecule normal presence in the myocyte). This realizes under significant inflammation or immune response transgene product is not had, although product may be a kind of neoantigen (neoantigen).

Significant especially with the transgenosis expression stability that the inventive method obtains. Do not wish to be bound by any theory, it is believed that this stability is due to when there is no helper virus or other external source accessory molecule, due to the AAV proviral DNA is very low with chromosomal integration efficiency in the myocyte. Some observed results have been supported this hypothesis. Described in hereinafter example, the viral genome of double-stranded attachment form when being analyzed, the Hirt extract could not be detected. The total DNA of cell is carried out Southern analyze, result shows, a discontinuous band is arranged when the enzymic digestion of two broken sites is arranged in the AAV carrier, and the enzymic digestion that there is no site in viral genome together-there is no discontinuous band during DNA.

Other DNA analysis lays particular emphasis on formation and the structure thereof of concatermer. The research of in the past cracking performance AAV being infected shows, the rAAV attachment copy by head to head or tail the tail concatermer is carried out, and the latent infection that causes provirus to be integrated realizes that to the tail tandem [54:316-329 (1990) for K.I.Berns, Microbiol.Rev. by head; J.-D.Tratschin etc., Mol.Cell.Biol., 5:3251-3260 (1985); N.Muzcyzska, Current Topics in Microbiology and Immunology, 158:97-129 (1993); S.K.McLauglin etc., J.Virol., 62:1963-1973 (1988)]. The data that this paper lists proved with the myocyte of rAAV carrier of the present invention transduction and contained the head that comprises two kinds of variable disappearances of the ITR concatermer to the tail arranged in series, and this is consistent with the transduction mechanism that relates to the integration of rAAV provirus.

The joint that the rAAV concatermer is produced carries out sequence analysis and shows that the disappearance of two kinds of ITR is consistent but can change. FISH (FISH) analyze with 20 karyons in approximately 1 have the result of single integration site to conform to, and Southern the analysis showed that each myofibrillar diploid gene group on average has a provirus genome. These discoveries show together, and concatermer is average minimum comprises 10 provirus genomes.

Another advantage of the inventive method is not have inflammation (this makes us being surprised) behind the carrier that gives therapeutic dosage.For example, lacZ AAV vector injection is gone into the C57BL/6 mouse does not have the humoral immunoresponse(HI) of appearance to the intestinal bacteria beta-galactosidase enzymes, has produced active anti--beta-galactosidase enzymes antibody although inject these animals of skeletal muscle back at the lacZ adenovirus carrier.Therefore, when using the carrier of non-auxiliary virus AAV, controlled to genetically modified immunne response according to the inventive method.Transgenic method in this and the prior art is diametrically opposite; these transgenic methods for example are the method [J.A.Wolff etc. that adopt naked plasmid dna; Science; 247:1465-1468 (1990)]; or can cause adenovirus mediated gene therapy method [] S.K.Tripathy that the transgenosis strong immunization is replied etc. usually; Nat.Med., 2:545-550 (1996)].Therefore, method of the present invention obviously is better than other genes delivery system, particularly needs aspect the chronic disease of repetitively administered in treatment.

I. reorganization AAV

Adopted in the inventive method and carried selected genetically modified AAV recombinant vectors.Except that transgenosis, carrier also contains the regulating and controlling sequence that the control transgenosis is expressed in host cell (as the myocyte).

Many rAAV carriers are well known by persons skilled in the art, so the present invention is not limited to any specific rAAV carrier.For example, non-limiting examples of suitable AV carrier and preparation method thereof is in U.S. Patent No. 5,252,479; 5,139,941; Describe to some extent among international patent application No.WO94/13788 and the WO93/24641.A kind of carrier of special needs is described below.

The A.AAV sequence

At present, a kind of preferable rAAV lack all viral open reading frame (ORF) and only kept 5 of cis acting ' and 3 ' inverted terminal repeat sequence (ITR) [for example referring to, B.J.Carter, at " Handbook ofParvoviruse ", P.Tijsser edits, CRC Press, pp.155-168 (1990)].Therefore, lacked the sequence of coding rep and cap polypeptide.The long approximately 143bp of AAAV ITR.Comprise 5 of basic all ITR ' and 3 ' sequence although carrier should adopt, those skilled in the art will be understood that can be to these sequences do little modifications to a certain extent.The ability of revising these ITR sequences and keeping its biological function is that those skilled in the art possess.For example referring to " Molecular Cloning.A Laboratory Manual. " such as Sambrook, the 2nd edition, Cold Spring Harbor Laboratory, the textbook of New York (1989).

AAV ITR sequence can obtain from any known AAV (comprise and determine people AAV type at present).The selection of AAV type does not wish to limit the present invention.All kinds of AAV (comprising the 1-4 type) can obtain from American type culture collection (ATCC), or ask each commercial and industrial source to provide.Equally, the AAV of known other animal of infection also can be used in the carrier that the inventive method adopts.In the embodiment that the present invention proposes, for simplicity, employing be AAV-2.Specifically, 5 of AAV-2 ' with selected transgenic sequence of 3 ' AAV ITR sequence side joint and relevant controlling element (as described below).

B. transgenosis

The transgenic sequence that contains in the rAAV carrier is the nucleotide sequence of allos in the AAV sequence, its can encode interested RNA or polypeptide.Transgenosis is connected with the controlling element operability in the mode that allows transgenosis to express in the myocyte.

The composition of transgenic sequence depends on the purposes of gained carrier.For example, a class transgenic sequence is included in the reporter gene that produces detectable signal when expressing.This report sequence includes but is not limited to intestinal bacteria beta-galactosidase enzymes (LacZ) cDNA, alkaline phosphatase gene and green fluorescence protein gene.When these sequences linked with the controlling element that drives its expression, they provided by the detectable signal of conventional means (as ultraviolet wavelength absorption, visible colour-change etc.).This genetically modified expression can be used in the methods such as cell quantitative assay, cell evaluation.

Better transgenic sequence comprises the therapeutic genes of the required gene product of encoding.These therapeutic nucleic acids sequences are the energy coded product usually, can substitute or proofread and correct heredity or non-genetic gene defective when this product gives the patient in vivo or in vitro and maybe can treat living (epigenetic) disease in back.

Method of the present invention (being about to transgenosis is delivered among the myocyte) is particularly suitable for being used in combination with secretion human cytokines (as factor IX (being used for the treatment of hemophilia), lipophorin (Apo) E (being used for the treatment of atherosclerosis)).Yet those skilled in the art also can select other therapeutic gene product (especially excretory therapeutic gene product).But the example of genes encoding excretory and/or diffusing products includes but is not limited to cytokine, somatomedin, hormone, differentiation factor etc., for example beta-interferon (β-IFN), erythropoietin (epo), Regular Insulin, tethelin (GH) and Rat parathyroid hormone 1-34 (PTH).These genes can be used to treat all kinds of diseases, comprise multiple sclerosis and cancer (β-IFN), anaemia (epo), diabetes (Regular Insulin), (GH) of short and small stature and osteoporosis (PTH).Method of the present invention also is suitable for the non-secretory product of genes encoding is delivered in the muscle.For example, estimate that method of the present invention can be used to treat muscle nutrition, method is that the rAAV by the inventive method sends the bad gene of nutrient protein [for example referring to, C.C.Lee etc., Nuature, 349:334-336 (1991)].Genetically modified selection can not be thought a limiting factor of the present invention, because this selection is those skilled in the art's capabilities.

C. the controlling element in the carrier

Except AAV ITR sequence and transgenosis, carrier comprises that also driving transgenosis expresses necessary controlling element in the myocyte of transduction.Therefore, wish that carrier contains selected promotor and the enhanser (if desired) that is connected with transgeneic procedure, they and transgenosis are all between the AAV of carrier ITR sequence.

Promotor and enhanser (if desired) can be selected according to usual manner, and this is not limited to carrier itself.Useful promotor can be to control the constitutive promoter of transgene expression or (induction type) promotor of adjusting.For example, the ideal promotor is cytomegalovirus immediate early promoter/enhanser [for example referring to, Boshart etc., Cell, 41:521-530 (1985)].Other ideal promotor includes but is not limited to Rous sarcoma virus LTR promotor/enhanser and induction type mouse metallothionein promoter.Those skilled in the art also can select some other promotor/enhancer sequence.

Wish that carrier also contains the nucleotide sequence that influential transgenosis is transcribed or translated, comprise sequence that the effective polyadenylation desired signal of transcript is provided and intron with functional donor splicing site and acceptor site.In typical carriers of the present invention, the common polyadenylation sequence of employing obtains from papovavirus SV-40.The polyadenylation sequence is inserted in behind the transgenic sequence in the carrier and before 3 ' AAV ITR sequence usually.Can also obtain common intron sequences from SV-40, it is called the SV-40T intron sequences.The required selection of components of these and other control or reinforcing gene expression is conventional, and many such sequences are [for example referring to, Sam Brook etc., and the reference of wherein quoting] known to those skilled in the art.

For the ease of the description of this paper, the combination of transgenosis, promotor/enhanser and other controlling element is called " minigene (minigene) ".As mentioned above, the minigene side joint 5 ' and 3 ' AAV ITR sequence.After instruction of the present invention was provided, those skilled in the art were easy to design this minigene.

An example (being AAV.CMVLacZ) that has provided rAAV in the following examples with and in the methods of the invention purposes.As shown in Figure 1, the rAAV of this demonstration contains 5 ' AAV ITR, CMV promotor, SV-40 intron, LacZ transgenosis, SV-40 polyadenylation sequence and 3 ' AAV ITR.Yet as mentioned above, method of the present invention is not limited to the use of any concrete rAAV.

The preparation of D.rAAV

According to having the material of publishing and describing in the past, the sequence that adopts when making up the rAAV that is used for the inventive method can obtain from commercial or science source.These materials also can from individual patient or recombinant molecule cloning process known with those skilled in the art and that put into practice accepted standard makes and screening obtains.Any variation (comprising sequence deletion, insertion and other sudden change) of the existing nucleotide sequence that is used to produce the rAAV carrier also available standards method is produced.

The assembling of rAAV (sequence, transgenosis and other carrier element that comprise AAV) can adopt ordinary method to realize.A kind of method of special hope is at K.J.Fisher etc., and J.Virol. describes among the 70:520-532 (in January, 1996) to some extent, and it is for referencial use that this partial content is included this paper in.Yet, other suitable method comprises that also the cDNA clone is (as textbook [Sambrook etc., the method of describing as mentioned above]), with AAV genomic overlapping oligonucleotide sequence, polymerase chain reaction and other any suitable method that required nucleotide sequence can be provided.In suitable, can adopt the transfection and the cotransfection method of standard, with propagation rAAV virus in the presence of helper virus (for example using the adenovirus of the E1 disappearance of CaPO4 transfection method), those skilled in the art are easy to this is made one's options.Can be used for other ordinary method of the present invention comprises the virus genomic homologous recombination of AAV, makes the viral method that grows up to plaque and measured signal generation on the layer of agar shop etc.

Hope is carried out adenovirus or the wild-type AAV of purifying to remove any pollution to the rAAV that produces.Special wish the purification scheme that adopts at K.J.Fisher etc., J.Virol., 70 (1): describe to some extent among the 520-532 (in January, 1996), it is for referencial use that this partial content is included this paper in.Yet those skilled in the art also are easy to select other suitable purification process.

II. treatment is used

In case obtain to contain required genetically modified rAAV, can directly give animal muscle with carrier.An advantage of the inventive method is more to be particularly suitable for as the position that produces secretion property treatment product (as factor IX or lipophorin (Apo) E) than other position at muscle.Perhaps, method of the present invention can be used to the non-secretory gene product is delivered among the myocyte.

RAAV carrier of the present invention should be suspended in physiologically acceptable solution or pharmaceutically acceptable carrier or the delivery vector and give the patient.A kind of suitable carriers is a Sterile Saline.For this purpose, also can adopt well known to those skilled in the art, known other water-based or non-aqueous isotonic sterile injection liquid and water-based or the non-aqueous sterile suspension that can be used as pharmaceutically acceptable carrier.

RAAV carrier of the present invention should give q.s, so that selected transgenosis is integrated and expression, thereby obtains result of treatment, does not have negative effect improperly, and medically acceptable physiological effect is arranged, and the dosage of carrier is that the medical field technician can determine.In a preferred embodiment, rAAV directly injects cardiac muscle, skeletal muscle or unstriated muscle.Those skilled in the art should be understood that also method of the present invention also can adopt other medication (as intravenously or intra-arterial injection), as long as rAAV target myocyte.

RAAV carrier dosage depends primarily on following factor: disease to be treated, selected transgenosis, patient's age, body weight and healthy state are for example arranged, therefore have different dosage for different patients.Think the treatment significant quantity of rAAV of the present invention between 1-50 mL of saline solution, every milliliter of this solution contains has an appointment 1 * 10 ⁸To 1 * 10 ¹¹RAAV carrier granule of the present invention.Wish that each dosage contains at least 10 ⁹The rAAV particle.Better human dosage is that about 1-20 milliliter has above-mentioned concentration salt brine solution.Can detect selected genetically modified expression level by biological test, to determine approach, dosage or the frequency of administration.The rAAV administration can repeat as required.

The embodiment that hereinafter lists has described the preparation carrier and has implemented the preferred approach of the inventive method.These embodiment only are intended for demonstration, and they do not limit the scope of the invention.

The generation of embodiment 1-AAV.CMVLacZ

Make a reorganization AAV (rAAV), wherein rep and cap gene are replaced by minigene (AAV.CMVLacZ), and this minigene can be expressed the intestinal bacteria beta-galactosidase enzymes under the control of CMV promotor.Make AAV.CMVLacZ[R.Samulski etc., J.Virol., 61 (10): 3096-3101 (1987) with the psub201 cis acting plasmid pAAV.CMVLacZ that obtains that derives].In brief, plasmid transfection is gone into to infect [K.J.Fisher etc., J in 293 cells of adenovirus of E1 disappearance, Virol., 70:520-532 (1996)], AAV rep and cap function provide [R.Samulski etc. by trans-acting plasmid pAAV/Ad, J.Virol,, 63:3822-3826 (1989)].As K.J.Fisher etc., J, Virol., described in the 70:520-532 (1996), every milliliter of genome copy number of each batch of titration AAV.CMVLacZ carrier product.

AAV.CMVLacZ genome (4883bp) from 5 ' to 3 ' structure comprise:

5 ' AAV ITR (bp-1-173) is by the Nucleotide 365-538 with pAV2[SEQ ID NO:1] be that template obtains [CA.Laughlin etc., Gene, 23:65-73 (1983)] with PCR;

CMV early stage immediately enhancers/promoters [Boshart etc., Cell, 41:521-530 (1985); The Nucleotide 563-1157 of SEQ IDNO:1],

SV40 intron (the Nucleotide 1178-1179 of SEQ ID NO:1),

Intestinal bacteria lacZ cDNA (the Nucleotide 1356-4827 among the SEQ ID NO:1),

SV40 polyadenylation signal (237BamH I-Bcl I the restriction fragment that contains the fracture/polyadenylation signal of early stage and late transcription unit; Nucleotide 4839-5037 among the SEQ ID NO:1) and

3 ' AAV ITR, the SnaB that from pAV2, obtains I-Bgl II fragment (the Nucleotide 5053-5221 among the SEQ ID NO:1).

Referring to Fig. 1, two BamH I sites are arranged in the double-stranded carrier sequence.First is arranged in SV40 intron bp875 place, second bp4469 place that is positioned between lacZ DNA and SV40 polyadenylation signal.Therefore digest the fragment that double-stranded sequence will discharge long 3595bp with the BamH I.Also show the cDNA probe location that is used for detecting inner BamH I fragment and total length carrier among the figure.

With standard method purifying rAAV.CMVLacZ virus [for example referring to, K.F.Kozarsky etc., J.Biol.Chem., 269:13695-13702 (1994)].Following test is used for the rAAV stock of the following example, to guarantee not duplicate the adenovirus of competitive wild-type AAV and auxiliary type E1-disappearance.

With 293 cell inoculations to slot type slide glass (chamber slide), and with wild-type adenovirus, equal portions rAAV carrier stock coinfection.Infect after 20 hours, fixed cell is cultivated cell with the mouse monoclonal antibody (American Research Products) of anti-AAV capsid protein.Second antibody with coupling FITC detects antigen-antibody complex.Positive signal is designated as an infectious AAV unit.Infect 293 cells with equal portions rAAV carrier stock, and expression is dyeed to the alkaline phosphatase reporter, to measure the auxiliary type adenovirus of polluting.The auxiliary type adenovirus is the E1 absence type, and it contains the placental alkaline phosphatase cDNA under the control of CMV promoter transcription.In highly purified rAAV prepared product, do not detected and duplicated emulative wild-type AAV or auxiliary adenovirus.

The embodiment 2-rAAV skeletal muscle of stably transduceing in vivo

Make rAAV with and not with adenovirus (purpose is the to strengthen transduction) administration of E2a-disappearance.Animal experiment obtains the approval of the Institutional Animal Care and Use Committee (IACUC) of Wistar Institute.

In brief, (Maine) anesthesia is cut 1cm with lower limb then for Jackson Laboratories, Bar Harbor to make female C57BL/6. mouse in 5 ages in week at peritoneal injection ketamine (70mg/kg) and xylazine (10mg/kg).With 25 μ l HEPES buffer saline (HBS, pH7.8) rAAV.CMVLacZ (1 * 10 in ⁹Or before injection, added adenovirus E2a mutant strain d1802 (5 * 10 the vector gene group), ¹⁰A ₂₆₀Particle, 1 * 10 ⁸Pfu) rAAV.CMVLacZ of [S.A.Rice and D.L.Klessing, J.Virol., 56:767-778 (1985)] goes in each leg tibialis anterior muscle (tibialis anterior muscle) through the Hamilton injector to inject.Otch is sewed up with 4-0 Vicryl suture.In order to analyze genetically modified expression, each time point cuts open the inspection animal after injection, downcuts the muscle of injecting with scalpel.Tissue is placed in the OTC embedding compound (embedding compound), and quick-frozen is 7 seconds in the iso-pentane of cooled with liquid nitrogen, moves in the liquid nitrogen immediately.The tissue of analyzing each time point provides minimum 6 injection sites (that is the sample of the both sides of at least 3 animals).

In order to carry out tissue chemical analysis, refrigerated muscle is horizontally divided into two parts of equal moieties, organize the cross section thereby produce.To two moiety continuous tissue sections (cutting) to 6 μ m.In order to carry out X-gal tissue chemical analysis, section is fixed in the 0.5% glutaraldehyde PBS solution of firm preparation, and as K.J.Fisher etc., J.Virol., the described such mensuration betagalactosidase activity that dyes of 70:520-532 (1996).Section is redyed in neutral red solution and sealing.

When with adenovirus during as the helper virus of rAAV, X-gal tissue chemical analysis shows the 17th day high-caliber myofiber transduction, and with significant inflammation.Yet, it is shocking that having accepted not, the animal of auxiliary adenovirus rAAV has shown above the being seen transduction level of adenovirus animal is arranged.These high levels can continue 180 days and obviously not disappear.

Embodiment 3-efficiently rAAV genome conformity thing be brachymemma the head to the tail concatermer

In order to analyze the genomic molecularity of stabilization rAAV, to as mentioned above in the injection mouse DNA of gained skeletal muscle carry out the Southern engram analysis.Consider to adopt two kinds of models: the rAAV genome exists with the double-stranded genome of attachment (those as forming when the lytic infection), or the integration provirus form of similar latent infection continues to exist.

In brief, on selected time point, from mouse muscle, separate obtaining low-molecular-weight dna (Hirt) (the A part sees below) and high molecular genomic dna (the B part sees below).With the DNA sample dissolution on 1% sepharose, by electrophoretic transfer to nylon membrane (Hybond-N, Amersham) on.Make trace and isolated from lacZcDNA, usefulness ³²The restriction fragment hybridization of P-dCTP random primer labelling.

A. the double-stranded genomic detection of attachment

In order to detect the rAAV genome of nonconformity form, with its with corresponding to probe sequence shown in Figure 1 ³²The cDNA of P-mark is hybridized, and has analyzed the Hirt extract of transduction muscle DNA.Hirt DNA sample (15 μ l are equivalent to the 15mg tissue) is that extracting goes out from the muscle of injecting the 8th, 17, the 30 and 64 day results in back.

The DNA that the clone that infects with rAAV in the presence of adenovirus is cultivated gained analyzes.To the analysis revealed of this clone Hirt extract, the monomer of single stranded form virus and double chain form virus all exists.Yet only showing with the muscle Hirt extract of rAAV transduction that the strand genome that produced in the 8th day was reduced at the 64th day can not detected level.In the Hirt extract, never detect the rAAV of double chain form, even when filter membrane over-exposure (over-exposed).This hint, strand rAAV genome is transferred in the Skeletal Muscle Cell effectively; But it is not transformed into the attachment of transcriptional activity form.

B. the detection of the proviral DNA of Zheng Heing and specificity analysis

In order to detect the proviral DNA of integration, the total DNA of gained cell carries out other hybridization research in the transduction Skeletal Muscle Cell when injecting back 64 days.With BamH I or Hind III (not cutting the Restriction Enzyme of proviral DNA) digested genomic dna (10 μ gm are equivalent to 18 μ g tissue).As expected, Hind III digestion gel component and with the virus-specific probe hybridization after produced a band.Yet, in being used in provirus, during the BamH I digested genomic dna of two places cuttings, detecting the discontinuous band that 3.6kb estimates size, its abundance is a nearly provirus genome in each diploid host cell gene group.

Identify the proviral structure of integrating with pcr analysis, to describe the possible mechanism of persistence.In the past the research of wild-type and recombinant type AAV was pointed out that in the cleavage stages and latency stage of viral life cycle, the approach that duplicates of DNA was different.Specifically, when having helper virus to exist, AAV by synthetic head to head or tail the mechanism of tail concatermer is duplicated the replicative intermediate that has formed dimerization.This opposite during with latent infection, the genomic characteristics of the provirus of integrating during latent infection are with head coda gene prescription formula to be arranged.

Genomic dna to Skeletal Muscle Cell carries out pcr analysis, the joint between amplification AAV genome concatermer.According to the rAAV of integration form normally head the observations of tail concatermer has been designed a kind of PCR method of integrating rAAV that detects.Specifically, the synthetic oligonucleotide primer thing optionally increases across two monomeric heads of AAV.CMVLacZ genome to the tail joint PCR.Sense strand primer 005 (5 '-ATAAGCTGCAA TAAAC AAGT-3 '; SEQ ID NO:4) corresponding to the bp4584-4603 in SV40 polyadenylation signal district.Antisense strand primer 013 (5 '-CATGGT AATAG CGATG ACTA-3 '; SEQ IDNO:2) corresponding to the bp497-478 of CMV promotor, and antisense strand primer 017 (5 '-GCTCT GCTTATATAG ACCTC-3 '; SEQ ID NO:3) corresponding to the bp700-680 of CMV promotor.If ITR is kept perfectly, oligonucleotide 005+013[SEQ ID NO:2 then] should amplify the fragment of 797bp, and oligonucleotide 005 and 017[SEQ ID NO:3] should amplify the primer of 1000bp.What highlight is that the expectation size of PCR product is the basis that is assumed to be that contains two ITR copies with the provirus joint.Therefore, amplification is that the size of the PCR product that produces of little joint can be correspondingly less than two copies.

The genomic dna template of 100ng and the primer concentration of 0.5 μ M are adopted in the PCR reaction.The thermal cycling process is 94 ℃ 1 minute, 52 ℃ 1 minute and 72 ℃ 1 minute 30 seconds, circulates 35 times; 94 ℃ of denaturing steps in the circulation are 2 minutes for the first time, and 72 ℃ of extensions of last round-robin step is 10 minutes.The PCR product is analyzed with agarose gel electrophoresis.

As mentioned above, from inject the AAV.CMVLacZ transduction muscle of back in the time of the 64th day, separate obtaining genomic dna, carry out the PCR reaction.Genomic dna with the muscle of having injected Hepes buffer saline (HBS) contrasts as negative PCR.Should cross over head to head or tail during adopting, not detected amplified production (data not shown) the primer of tail joint.Yet, when analyzing the myocyte DNA of AAV.CMVLacZ transduction with oligonucleotide 005 and 013 and 005 and 017, detect a spot, this have with the specificity head to tail concatermer faciation symbol (Fig. 2 A and 2B).

In addition, carry out the PCR reaction with the genomic dna that contains the clone of integrating AAV.CMVLacZ.Measured these clones' provirus structure with the Southern engram analysis.Identify three clones (10-3.AV5,10-3.AV6 and 10-3.AV18), they each all contain the monomer copy that the AAV.CMVLacZ of at least two integration arranges the tail mode with head.According to the size (with the product that primer sets 005-013 obtains 720bp, using primer sets 005-017 to obtain the product of 930bp) of PCR product, two clones (10-3.AV5 and 10-3.AV6) contain the AAV ITR of 1.5 copies similarly in the joint.Another clone 10-3.AV18 contains a large amount of disappearances that comprise AAV ITR, has produced the product of 320bp with primer sets 005-013, and has produced the product of 500bp with primer sets 005-017.Another clone 10-3.AV9 contains a monomer copy (according to the Southern engram analysis) of integrating AAV.CMVLacZ, and it seems that the disappearance by the PCR product is confirmed.

Therefore, shown less than entire header the big or small clear band of tail concatermer expectation selecting the analytical results of stablizing the cell line dna of the rAAV infection of transduceing for use.

D. structural analysis

By the subclone of PCR reactant (Fig. 3), carry out restriction analysis (Fig. 4 A-4G) then, the provirus joint that reclaims from the bone musculus dna is carried out detailed structural analysis.Specifically, the PCR product of above-mentioned gained BL.11 myocyte sample is directly connected in the commercial plasmid pCR II of buying, wherein the inset side joint EcoR I site.Transform the commercial competence bacterial strain TOP10F ' that buys with ligation.In fact, this step has produced the plasmid library of PCR product.Make that this library is dull and stereotyped cultivates, bed board density is enough to provide abundant isolating bacterium colony, be covered with nylon membrane and with corresponding to CMV promotor/enhanser ³²The fragment that-P mark is crossed is hybridized and is screened.Make the positive colony of inferring cultivate grow overnight in (2 milliliters) on a small scale.

Extracting goes out 6 typical case clones' plasmid DNA from cultivate on a small scale, digests with the EcoR I then and discharges whole PCR product, or digest with SnaB I (as diagnostic markers).Should discharge the 306bp fragment (SnaB I 476 to SnaB I 782) of crossing over the CMV promotor with the digestion of SnaB I.Decide corresponding to the rearrangement that the release of second fragment (SnaB I 142 to SnaB I 476) of ITR joint takes place when forming concatermer, so magnitude range is that 334bp (2 complete ITR copies) is to 0bp (if ITR lacks).

To think that containing the 1.5 PCR products that copy the clone 10-3.AV5 of AAV ITR (10-3.AV5) also is cloned in the pCR II, and use specified enzymic digestion.This sample is as the positive control of diagnostic SnaB I digestion.Digest the secondary doublet band that this sample can correctly discharge the long 500bp of PCR fragment peace treaty of 730bp with the EcoR I.Think that this secondary strap is because secondary structure results from 1.5 copies of AAV ITR when bacterium is duplicated.Can from the CMV promotor, discharge diagnostic 306bp fragment and corresponding to the 250bp fragment of ITR joint with SnaB I digestion positive control.

Test with EcoR I and 6 clones of SnaB I digestion shows, all has the disappearance that is uneven in length in the joint of all recovery, and is confined to the ITR of joint to a great extent.Sequential analysis shows that further the great majority disappearance has been crossed over two portions ITR of joint, but does not relate to the viral DNA that adjoins.

E. fluorescence in situ hybridization (FISH) is analyzed

On the skeletal muscle freezing microtome section, carry out fish analysis, to determine the distribution of proviral DNA in the tissue of injecting.The muscle fritter of the treatment or the 4-5mm of control mice is embedded among the OTC, and in the liquid iso-pentane of cooled with liquid nitrogen quick freezing.The freezing microtome section that 10 μ are thick switches on the cryomicroscope.Settle, fix (Histochoice) and carry out the in situ hybridization processing and be convenient to fluorometric analysis with the existing method of describing [E.Gussoni etc., Nat.Biotech., 14:1012-1016 (1996)].The betagalactosidase activity staining analysis is carried out in the section of adjoining, to determine the lacZ positive region in the fascicula.

For quantitative assay FISH signal, check lacZ positive region (determining) being equipped with on the Nikon microphot FxA microscope that falls to penetrating fluorescence by dying the betagalactosidase activity that bag analysis adjoins section.The single myofibrillar sum of lacZ positive region in the counting section under the pattern that differs of standard.Then, under fluorescent microscope, check the same area with suitable isothiocyanic acid rhodamine colour filter bag (filter package).Record demonstrates the painted muscle corpuscle number of point-like.Under the pattern of differing, check each male karyon, to determine that it is from myofiber.For contrast, with the same manner inspection and the negative zone of quantitative assay lacZ (lacking the zone of betagalactosidase activity or the flesh section of simulation transfection in the same section).

For focusing microscope, the Leica confocal laser microscope that Krypton-argon laser apparatus (TSC and Voxel View Silicon central authorities graphics workstation) is housed (Leica Lasertechnik, GmbH), go up oil-immersion objective (100X) and observe section down.View image under the rhodamine passage differs down at differential interference subsequently and observes, to confirm that fluorescent signal is arranged in muscle corpuscle.Differential is differed with fluorescent image then and stack gradually on the TCS central workstation, and transfer to and carry out image processing in the Silicon graphics workstation.Preserving processing back image also prints with Photoshop software.

Perhaps, the betagalactosidase activity of serial section is measured in dyeing, with the myofiber of definite transgene expression, and makes section and biotinylation provirus probe hybridization, to determine the genomic distributing position of provirus.In the myofiber karyon of some expression beta-galactosidase enzymess, detected discontinuous fluorescent signal.The investigation result of three serial section is shown have 53 hybridization is arranged in the fiber finer karyon that 1006 are expressed beta-galactosidase enzymess, and none there is hybridization in 377 myofiber nucleus of not expressing beta-galactosidase enzymes.Do not detecting hybridization (data are unlisted) in the infected animals tissue.

Detect virus genomic ability with FISH and provide another kind of yardstick for analysis.The contained all cells nuclear of the myofiber of expressing beta-galactosidase enzymes 5% in detected single hybridization point.Because the sensitivity limited (especially to the target sequence less than 12kb) of this method, this may cross the karyon [B.J.Trask, Trends Genet., 7:149-154 (1991)] that transduction has been estimated in the lowland.The hint of fish analysis is interesting.There is a provirus locus to have (recording) in each diploid sarcoplast genome, and proves that 5% nucleic acid has carried the genomic FISH result of AAV, indicated that concatermer on average comprises 10 provirus genomes at least through Southern.These betagalactosidase activities that studies show that out have extended beyond the nucleus position of carrier transduction far away, illustrate that examining the zone has extended 10 μ m at least.This and former result of study (have been put down in writing nuclear district [H.M.Blau etc., Adv.Exp.Med.﹠amp that cytostolic albumen has extension; Biol., 280:167-172 (1990)]) be consistent.Owing to some reasons, it is very important that the transgene expression nuclear district of extending in the myofiber synplasm structure uses gene therapy.In the protein distribution was subjected to envelope barrier constraint synplasm still less, the net production (net yield) of the recombinant protein that the transduction activity forms may be higher.And, to express in the carrier system of many times of recombinant proteins (if any those albumen of inducible promoter) at needs, native system also has advantage [J.R.Howe etc., J.Biol.Chem., 270:14168-14174 (1995); V.M.Rivera etc., Nat.Med., 2:1028-1032 (1996)].In muscle, because the extension network of overlapping region, the co expression of recombinant protein does not need to transduce jointly in the karyon.

Embodiment 4-when with the rAAV that does not contain helper virus with the gene targeted delivery to muscle the time, it is minimum that the immunne response that transgenosis causes is reduced to

Occurred the destructive immunne response of the beta-galactosidase enzymes of expressing at adenovirus carrier in the myofiber in view of former studies have shown that, therefore the lacZ expression stability is wonderful in the myocyte that the rAAV carrier that contains lacZ that does not have helper virus is realized.By the serum level of the anti-beta-galactosidase enzymes antibody of Western assay determination, further study the transgenosis specific immune response.

(Jackson Laboratories, Bar Harbor get blood and collect serum in ME) for C57BL/6 that dissects after 30 days from injecting virus and ROSA-26 mouse.ROSA-26 is the transgenic lines mouse of carrying intestinal bacteria beta-galactosidase enzymes cDNA, and growing up in 129 mouse backgrounds forms.Also from C57BL/6 mouse results serum, this mouse has been accepted reorganization LacZ adenovirus (H5.010CMVlacz, 5 * 10 in addition ⁸Pfu is in 25 μ l HBS) or the reorganization AAV.CMVLacZ (as mentioned above) intramuscularly.Two carriers all give expression to the intestinal bacteria beta-galactosidase enzymes from the minigene that CMV drives.Intestinal bacteria (Sigma) the beta-galactosidase enzymes equal portions (5 μ g) of purifying are dissolved in 10% sds page (5mg/ swimming lane), and by electrophoretic transfer to the Nitrocellulose film (Hybond-ECL, Amersham) on.Making trace and blotto[5% nonfat milk, 50mM Tris-HCl (pH8.0), 2mM CaCl and 0.05% tween 20] room temperature cultivated 2 hours, the site that may exist with blocking-up.Downcutting each swimming lane cultivated 1 hour with serum (being diluted among the blotto with 1: 200) room temperature.Add goat anti-mouse horseradish peroxidase thing, carry out ECL then and detect (Amersham), mark the position of antigen-antibody complex.The swimming lane that downcuts is ressembled on the polyester film, added ECL reagent then and and carry out thin film recording.

To not have the H5.010CMVlacZ E1 intramuscularly of adenovirus in the C57BL/6 mice skeletal, can cause a large amount of accumulation beta-galactosidase enzymes antibody in the serum, and this can not take place in the identical transgenic animal (beta-galactosidase enzymes is had immunotolerance) of the MHC-of the lacZ gene that carries insertion.Clearly, behind intramuscularly AAV.CMVLacZ, C57BL/6 and lacZ transgenic animal all do not produce the antibody of anti-beta-galactosidase enzymes.

The comparative study of adenovirus and AAV carrier among the embodiment 5-myocyte

The biology of research reorganization AAV and the adenovirus mediated transgenosis that is oriented to the myocyte, the result shows that adenovirus (rather than AAV) has infected antigen presenting cell (APC), this cell has caused the immune response of waterfall type, causes in the destructive cell and humoral immunity.

Set up a test example, to determine that the host is to the reorganization AAV that is oriented to skeletal muscle and the concrete difference of adenoviral gene transfer acknowledge reaction.Purpose is in order to describe these carrier systems in biologically difference, and these carrier systems can produce the preferential immuno-stimulating at transgene product when recombinant adenoviral vector (but not being the AAV carrier) express transgenic product (being beta-galactosidase enzymes).

Usual method is that the AAV that will express lacZ is expelled in the right leg of mouse.Demonstrate in the above-described embodiments, can provide effective and stable genetic expression like this.In other test group, animals received rAAV and carrier and cell have carried out various combinations, and at the immunoreactive composition of adenovirus (Ad), Ad causes destroying cellular immunization and humoral immune reaction just with clearly.Estimate these experimental implementation then to the influence of the myofiber stability that moves into rAAV and measure the effect that other immune parameter is determined these experimental implementation.By the stability and the development of inflammation of transgene expression in the myofiber of estimating the AAV transduction, thereby can detect any interfering factors that causes anti-beta-galactosidase enzymes immunizing power in the myofiber.

The following conclusion, for this research and design four experimental group.With the method that is similar to the foregoing description 2 substantially (soon virus is suspended in the phosphate buffered saline(PBS) and is injected directly in the tibialis anterior muscle) virus injection is gone in the mouse body.After dissecting mouse, the muscle tissue quick freezing in the iso-pentane of cooled with liquid nitrogen and be cut into the thick section of 6 μ m, is collected serum sample simultaneously and drained inguinal lymph nodes and be used for immunity test.

From inguinal lymph nodes, gather in the crops lymphocyte, 6 hours of carrying out standard as described below substantially ⁵¹Chromium (Cr)-release test adopts different effector and target cell (C57SV, H-2 among the 200 μ l DMEM in 96 orifice plates of V-arrangement bottom ^b) ratio.With before the effector cell mixes, target cell with the adenovirus (AdALP) of expressing alkaline phosphatase or the stable retrovirus pLJ-lacZ infection of having transduceed and having expressed lacZ, is used 100 μ Ci's earlier again ⁵¹The Cr mark, each hole adopts 5 * 10 ³Individual cell.After cultivating 6 hours, counting 100 μ l supernatant liquor equal portions in gamma counter.The result of 1-3 group is provided among Fig. 5 A-5C.

Freezing microtome section (6 μ m) fixed with methyl alcohol and with anti--CD4 and anti--CD8 antibody staining.Carry out the cellular form Measurement and analysis, to determine CD8+ cell and the CD4+ cell count in each section.

Release of cytokines is measured basically according to mode hereinafter described and is carried out.AAV or 5 type adenovirus with beta-galactosidase enzymes, purifying stimulated lymphocyte 40 hours again.Measure the secretion of middle IL-10 of acellular supernatant liquor (100 μ l) and IFN-γ.After 72 hours with 8 hours ³H-thymidine (0.50 μ Ci/ hole) pulse measurement propagation situation.Four groups of results are provided among Fig. 6 A and the 6B.

Substantially carry out the neutralizing antibody test by following step.Cultivate mice serum sample 30 minutes with the deactivation complement, make the twice serial dilution from 1: 20 with DEME then for 56 ℃.(100 μ l) mixes with beta-galactosidase enzymes or 5 type adenovirus with each serum dilution.37 ℃ of cultivations added the DMEM that 100 μ l contain 20%FBS after at least 60 minutes in each hole.Fixed cell also dyes in the date afterwards and measures the expression of beta-galactosidase enzymes.All cells is not all dyed blueness when having serum sample.Four groups of results are provided among Fig. 6 A and the 6B.

The right leg of the 1st group of mouse has been accepted the AAV.CMVLacZ that makes as described in embodiment 1, and does not have other interfering factors.Single produced high-caliber, stable transgenosis (even can both see at the 28th day) and do not have lymphocyte and infiltrate with the AAV.CMVLacZ transduction.Do not detect CD8 T cell be activated (Fig. 5).Do not detect the CD4 of antigen-specific yet ⁺T cell [that is, virus or beta-galactosidase enzymes antigen (Fig. 6 A and 6B)].Do not produce the antibody (Fig. 7 A and 7B) of anti-beta-galactosidase enzymes or adenovirus.

The right leg of the 2nd group of mouse is accepted AAV.CMVLacZ, and left leg accepts to express the adenovirus (H5.010CMVlacZ) of lacZ.The purpose of this group is in order to determine whether the myofiber institute immune response at the Ad infection is (the proving by its biological impact to the opposite side leg of AAV.CMVLacZ transduction) of general.Clearly, adenovirus lacZ handles the immunne response of having induced anti-beta-galactosidase enzymes, causes AAV lacZ transduction fiber destroyed.This is not wondrous, this with CD4 and CD8 T cell all infiltrate in the leg of AAV transduction relevant, and with at adenovirus and the antigenic cytotoxic T lymphocyte activation of beta-galactosidase enzymes relevant (Fig. 8).Also observe in addition at antigenic specific, the activated cd4 t cell of AAV, Ad and beta-galactosidase enzymes with to the specific antibody of adenovirus and beta-galactosidase enzymes.

The right leg of the 3rd treated animal has been accepted the mixture of AAV.CMVlacZ and Ad Bgl II.Ad Bgl II is the adenovirus of the E1 disappearance of not express recombinant gene.The purpose of this group is in order to determine whether adenovirus can provide the auxiliaring effect that causes anti-AAV and lacZ immunizing power in this case.This does not have render transgenic to express forfeiture, oozes out but have in a large number at the CD8 T cell of virus antigen (rather than beta-galactosidase enzymes), and at the CD4 T cell of virus antigen be activated (Fig. 6 A-6B).As expected, produce the antibody of anti-adenovirus, but do not produced the antibody (Fig. 7 A-7B) of anti-beta-galactosidase enzymes.

The right leg of the 4th treated animal has been accepted AAV.CMVLacZ, uses from the antigen presenting cell adoptive transfer of natural animal acquisition and with adenovirus and in vitro infects.

In these animals, set up vigorous and effective immunne response (confirming) by transgene expression loss and CD8 and a large amount of infiltration of cd4 cell to beta-galactosidase enzymes.In this experiment, be activated (shown in Fig. 6 A-6B), and produced the antibody (shown in Fig. 7 A-7B) of anti--beta-galactosidase enzymes at the CD4 T cell of beta-galactosidase enzymes.

The rAAV carrier transduction that embodiment 6-will contain the purifying of factor IX is gone among the bone myocyte, treating useful horizontal expression F. IX, and can not cause that cytotoxic immune replys.

The data that provide in the present embodiment show that method of the present invention can provide the expression of the therapeutic genes (F. IX) of prolongation in immunocompetent individuality and in lacking the immunoincompetent individuality that the cytotoxic immune of transducer cell is replied.In addition, the protein level of gained is enough to reach result of treatment in immunoincompetent animal serum.Therefore, express, just can be used to the F. IX is delivered to the patient who suffers from this disease by the people F. IX that the rAAV carrier prolongs in no immunological competence patient (as suffering from the patient of the hemophilia B) myocyte.

A. the preparation of the rAAV of purifying

The rAAV carrier that is used for the interior experiment of style has down carried an expression cassette (sequence), this box contains people F. IX cDNA, this cDNA comprise be subjected to cytomegalovirus (CMV) immediate early gene promotor/enhanser and and the SV40 transcription termination signal transcribe the part of the intron I of control.Make up in the following manner and contain the expression cassette of side joint AAVITR sequence and the carrier that the AAV albumen coded sequence lacks fully.

With F. IX cis plasmid (pAAV-FIX) and trans-acting plasmid pAAV/Ad[A.W.Skulimowski and R.J.Samulski, Method.Mol.Genet., 7:7-12 (1995)] (this cell is as Fisher etc. for common transfection human embryo kidney (293) cell, J.Virol., the described adenovirus that infects the E1 disappearance of 70:520-532 (1996)), produce reorganization AAV.PAAV-FIX is from psub201[Skulimowski and Samulski, the same] obtain, it contains CMV promotor/enhanser, comprises the people F. IX encoding sequence [S.Kurachi etc. of 1.4kb fragment intron I, J.Biol.Chem., 270:5276-5281 (1995)] and SV40 polyadenylation signal, their side joints AAV ITR sequence.AAV rep and cap gene function provide by pAAV/Ad is trans.The disappearance E1 adenovirus contain beta-galactosidase enzymes (LacZ) or alkaline phosphatase (ALP) reporter gene, with spike rAAV stock by this helper virus potentially contaminated.At transfection ultrasonic ruptured cell after 48 hours, the rAAV particle that discharges with four CsCl density gradient centrifugations (as described in Fisher etc., the same) purifying.

Gained rAAV-F.IX particulate density is 1.37-1.40g/ml.By slot blot hybridization, adopt the pAAV-F. IX plasmid DNA standard substance that CMV promotor or intron I sequence had specific probe and concentration known, measure the titre of the rAAV-F. IX of purifying.HeLa cell by the transduction growth, with the specific ELISA of hF. IX being measured the hF. IX concentration [J.Walter etc. in the culture supernatant when infecting back 36 hours, Proc.Natl.Acad.Sci.USA, 93:3056-3061 (1996)], confirm the ability of rAAV-F. IX at external transducer cell.With rAAV-F. IX (10 ¹²-10 ¹³Genome/milliliter) is deposited in-79 ℃, HEPES buffer salt solution (pH7.8 comprises 5% glycerine).

When measuring alkaline phosphatase or beta-galactosidase enzymes (as described in (the same) such as Fisher), but find that the rAAV-F. IX of purifying does not have the adenovirus of detection limit to pollute usually when dyeing then by 293 cells of transduceing.Detect wild-type AAV per 10 ⁹Individual rAAV-F. IX genome has less than 1 infectious unit.The measuring method of wild-type AAV is as follows: will be grown in 293 cells on the slot type slide glass with the rAAV-F. IX co-infected of adenovirus and equal portions purifying, and fixing after infecting 24 hours, to carry out immunofluorescence dyeing.(MA) as first antibody, extent of dilution is that (DAKO Corporation, Carpinteria is CA) as second antibody for 1: 40 anti-mouse IgG for American Research Products, Belmont with the mouse monoclonal antibody of anti-AAV capsid protein.

B. rAAV is introduced in the skeletal muscle

The mouse kind of selecting intramuscularly rAAV for use be C57BL/6 (Charles Fiver Laboratories, Wilmington, MA) and B6,129, Ragl (Jackson Laboratories, Bar Harbor, Maine).Peritoneal injection ketamine (70mg/kg) and xylazine (10mg/kg) make 4-6 female mice anesthesia in age in week, and vertically cut the otch of long 1cm at lower limb.(in the HEPES buffer salt solution (pH7.8), each animal is 2 * 10 with the AAV-F. IX ¹¹Or 1 * 10 ¹⁰Individual vector gene group) goes in the tibialis anterior muscle (25 μ l) and quadriceps muscle of thigh of every leg with the Hamilton injector to inject.Sew up the incision with 4-0 Vicryl suture.Every 7 days fibre bundle behind socket of the eye (retro-orbital plexus), collect blood sample in the microhematocrit kapillary, measure hF. IX (hereinafter C part) in the blood plasma with ELISA.Measuring (hereinafter D part) and DNA analysis (hereinafter F part) for immunofluorescence dyeing, then is in selected time point execution animal and the muscle tissue of downcutting injection and not injecting.Tissue is placed in the OTC embedding compound, 7 seconds of quick freezing in the iso-pentane of cooled with liquid nitrogen, and transfer in the liquid nitrogen immediately.

C. detect people F. IX with ELISA

As described (the same) such as Walter, with the people F. IX antigen in the ELISA mensuration mice plasma.This ELISA method not with the cross reaction of mouse F. IX.All samples is done duplicate mensuration.Muscle is immersed in ultrasonication then in the phosphate buffered saline(PBS) (PBS) that contains leupeptin (0.5mg/ml), makes the albumen extract from mouse muscle through injection.Remove cell debris by microcentrifugation, with the hF. IX in the albumen extract of ELISA mensuration dilution in 1: 10.With the injection rAV.CMVLacZ muscle extract (seeing the foregoing description 1) as negative control.(Bio-Rad, Hercules CA) measure protein concentration with the BIORAD test.

D. immunofluorescence dyeing

In order to carry out the immunofluorescence dyeing of tissue slice, the freezing microtome section (6 μ m) of muscle tissue is fixed on 3% poly-many formaldehyde (PBS, pH7.4) in 15 minutes, PBS cleaned 5 minutes, in methyl alcohol, cultivated 10 minutes, it is inferior to give a baby a bath on the third day after its birth with PBS, is placed on then in the PBS/3% bovine serum albumin (BSA) and seals 1 hour.To cut into slices then with affinity purification, cultivate with the anti-people F. of the goat IX antibody (AffintiyBiologicals) of PBS/1%BSA 1: 1000 dilution and to spend the night.After in PBS/1%BSA, washing three times (each 10 minutes), apply second antibody (the anti-goat IgG of FITC-link coupled rabbit, DAKO Corporation is with PBS/1%BSA dilution in 1: 200) 90 minutes.After washing three times again in PBS/1%BSA, with the section of distilled water rinse, dry air is also used Fluoromount G sealing medium (Fisher Scientific) sealing.All incubation step are at room temperature carried out, except cultivating with first antibody (4 ℃).(Chemicon, Temecula CA) as first antibody (extent of dilution is 1: 500), when dyeing as second antibody, adopt same step with the anti-rabbit igg of FITC link coupled (DAKO Corporation) with the anti-people's collagen of rabbit IV when section.For co research, adopt simultaneously with FITC link coupled goat to resist-hF. IX antibody (Affinity Biologicals) and anti--collagen IV antibody, detect collagen IV-antibody complex with the anti-rabbit igg of rhodamine link coupled (Chemicon).The fluorescence microscopy microscopy adopts the NikonFXA microscope.

E. test the circulating antibody of anti-hF. IX

In the plasma sample of the C57BL/6 mouse of intramuscularly AAV-F. IX, whether there is anti-hF. IX antibody with the ELISA test.(1 μ g/ml joins with the 0.1M sodium bicarbonate personnel selection F. IX, and pH9.2) bag is by microtiter plate.Adopting bipartite diluting plasma sample (1: 16), is that (Zymed, San Francisco CA) detect anti-hF. IX antibody for 1: 2000 and anti-mouse IgG horseradish peroxidase with extent of dilution.Buffer condition is as described in (the same) such as Walter.Be diluted to ultimate density be 1 μ g/ml mono-clonal mouse anti-hF. IX (Boehringer Mannheim) light absorption value relatively, estimate anti-hF. IX level.As Dai etc., Proc.Natl.Acad.Sci.USA, described in the 92:1401-1405 (1995), prove the existence of anti-hF. IX with the Westen blotting, just use goat anti-mouse IgG antibody (BoehringerMannheim) with horseradish peroxidase as second antibody, thereby can detect hF. IX-antibody complex with ECL reagent (Amersham).The extent of dilution of mice plasma is 1: 500.

F.DNA analyzes

As Sambrook etc., Molecular Cloning:a Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, described among the NY (1989) about mammalian tissues, from the muscle tissue of injecting, separate obtaining genomic dna.As described in the embodiment of the present application 3, carry out PCR reaction, with the head of amplification rAAV tandem repetitive sequence to the tail joint.Forward primer 005[SEQ ID NO:4] annealing SV40 polyadenylation signal (bp8014-8033), reverse primer 013[SEQ ID NO:2] and 017[SEQ ID NO:3] in conjunction with CMV promotor (bp4625-4606 and 4828-4809).The PCR reaction adopts the 100ng genomic dna (comprising 1.5mM MgCl ₂The total reaction volume of 100 μ l in) and 0.5 μ M primer to 005/013 or 005/017.Behind initial denaturing step (94 ℃ 4 minutes), carry out 35 circulations of following process: 1 minute, 52 ℃ annealing of 94 ℃ of sex change are extended 90 seconds (being 10 minutes in the circulation the last time) for 1 minute, 72 ℃.Clone test kit (Invitrogen, San Diego, CA) clone PCR products (being used for dna sequence analysis) with T/A.With ³²That P-dCTP guides at random, have specific label probe to carry out the Southern blot hybridization to CMV promotor (be used for PCR fragment hybridization) or the intron I (being used for hybridization) that is present in the hF. IX of rAAV-F. IX with genome mouse DNA.

G.hF. the expression of results of IX in immunocompetent mouse

The intramuscularly of rAAV-hF. IX in immunocompetent C57BL/6 mouse, was put to death animal in back 1 month in injection.Albumen extract through injection muscles (tibialis anterior muscle and quadriceps muscle of thigh) is carried out ELISA measure, the result shows that every milligram of tissue has the hF. IX of 1.8-2.1ng to have (every milligram of albumen has 40-50ng hF. IX).Can confirm to have in the muscle tissue hF. IX to express by the immunofluorescence research on the tissue slice.

The intramuscularly of rAAV-hF. IX is gone in the immunocompetent C57BL/6 mouse body, and put to death animal in the time of back 3 months in injection.In the muscle of not injection, do not detect factor IX.In the muscle of having injected contrast (rAAV-lacZ), do not detect factor IX.In back 3 months C57BL/6 rabbit myofiber of injection, detected people F. IX and expressed that (each injection site has 3.3 * 10 ¹⁰The vector gene group; Magnification is 200 times).Notice that the F. IX does not exist only in the myofiber itself, and in interfibrous matter space, show accumulation.

Interesting is that this dyeing pattern is identical with testing finding (a matter space also is colored) with the polyclonal antibody of anti-people's collagen IV.(each injection site has 3.3 * 10 to injection rAAV-hF. IX ¹⁰Individual genome) can the choose antibody of collagen IV of muscle dyes well after 1 month.Having identified the collagen IV recently is a kind of conjugated protein [W.-F.Cheung etc., Proc.Natl.Acad.Sci.USA, 93:11068-11073 (1996)] of people F. IX.

Tentative experiment in the immunocompetent mouse proves that although have high-caliber transgenosis and stable hF. IX to express in the muscle of injection, can not detect with ELISA has the hF. of significant quantity IX in the circulation.See Fig. 8.Other experiment showed, that animal has produced the antibody of the anti-cyclicity foreign protein of high titre.For example, when test in the same plasma sample during anti-people F. IX antibody, can in the animal body that all were injected, observe the intensive antibody response rising in the 11st day after the injection.See Fig. 9.Use the Western engram analysis, find to continue to be present in experimental session at high-caliber circulating antibody.

This discovery is opposite with the experience of report before us, report in the past is to give different carrier (promptly expressing the adenovirus carrier of hF. IX) by different approach (being intravenous injection) to have produced different immunne response (promptly not triggering the formation of anti-hF. IX neutralizing antibody) [Walter etc., the same].

Yet it is very low to induce antibody to form required protein expression level.Western trace test is to a certain extent not as the ELISA sensitivity, but it has been put down in writing antibody titers and rose in the 18th day after injection and begin to increase.Therefore, in immunocompetent animal body, not detecting the expression of hF. IX when start time point is the biological results that intramuscular rAAV (as yet not) expresses, and is because due to the generation of anti-foreign protein antibody and do not detect the F. IX in the circulation subsequently.Yet serum antibody response is with uncorrelated at the cellular toxicity immunne response of transgene expression cell.In fact, on above-mentioned arbitrary tissue slice, all do not find inflammation and tissue injury widely, at H﹠amp; Do not find in the section of E staining analysis (data are unlisted) yet.This with will carry genetically modified recombinant adenovirus be injected into skeletal muscle cause the immunne response situation [Dai etc., the same; Y.Yang etc., Hum.Molec.Genet., 5:1703-1712 (1996); X.Xiao etc., J.Virol.70:8098-8018 (1996)] opposite.

H.hF. the expression of IX in the immunodeficiency type mouse

The AAV-F. IX is delivered in the muscle of Rag1 mouse (sporting in the recombinase activated gene 1 is homozygous).Therefore, these mouse in severe combined immunodeficiency (SCID) mouse, and can not produce sophisticated B or T cell on the first-class rank of function.Dosage in each animal is 2 * 10 ¹¹Individual rAAV-hF. IX vector gene group causes stably expressing in the mice plasma hF. IX.See Figure 10.In second week after injection, available ELISA at first detects people F. IX, and after this increases gradually.In 5-7 week after injection, the blood plasma level in all animals reaches the steady stage of F. IX treatment level, and 200-350ng hF. IX is arranged in every milliliter of mice plasma. keep this level at experimental session (injecting in back 4 months) always.When being injected into altogether 1 * 10 ¹⁰RAAV-hF. during IX vector gene group, expression will be hanged down 3-4 doubly, but has still reached treatment level (greater than 100ng/ml) for some animals.See Figure 11.These levels (4-7% of normal circulation level in the expression blood plasma) and show that method of the present invention is feasible for treatment hemophilia (a kind of with immune deficiency diseases associated) in therapeutic domain.

The I.DNA analytical results

6-8 week isolates genomic dna through the injection muscles tissue after injecting.By the digestion of EcoR V, from vector construct, discharge the 1.8kb fragment that comprises whole 1.4kb intron I sequence, can prove that the carrier DNA of importing exists.To the intron I have specific probe can with the hybridization of this fragment, but not with mouse DNA cross hybridization without the injection animal.Indigested DNA demonstrates hybridization signal in high-molecular-weight DNA.In addition, be used for increasing the head of reorganization AAV in the transducer cell to successfully increased this sequence of from the tissue (tibialis anterior muscle of immune deficiency and immunocompetent animal and quadriceps muscle of thigh) of AAV-F. IX transduction isolated muscle DNA of the PCR primer (Figure 12) of tail concatermer joint sequence.The PCR product is by showing with CMV promotor/enhanser there being specific probe Southern blot hybridization.Primer has produced 1.0kb and littler fragment to 005-013; Amplification has obtained 1.2kb and littler fragment to primer to 005-017.As expected, these PCR instead would not produce the clear band of above-mentioned size, but a series of amplified productions maximum as estimating, because exist coarse AAV genome to connect [S.K.McLaughlin etc., J.Virol.62:1963-1973 (1988)] in these tandem repetitive sequences.Coarse connection is because the ITR sequence of joint area has variable disappearance (dna sequencing of the PCR product through cloning can confirm that data are unlisted).

J.PCR research

Although the AAV genome can take place head to head and the arrangement [K.I.Berns of tail to tail during virus replication, Microbiol.Rev., 54:316-329 (1990)], but when latent infection head to the arrangement of tail more generally be integrated into the transducer cell chromosomal DNA in the relevant [S.K.McLaughlin etc. of AAV, J.Virol., 62:1963-1973 (1988); J.D.Tratschin etc., Mol.Cell Biol., 5:3251-3260 (1985); N.Muzcyka, Current Topics in Microbiology and Immunology, 158:97-129 (1992)].

The Southern trace data of the undigested DNA of myocyte of injection rAAV show that the 6th week existed with the high molecular weight material form from the rAAV DNA that host cell gene group DNA obtains after injection.The being seen more high-strength signal of restrictive DNA may be because due to the screen unlocking effect when isolating fragment from a large amount of genomic dnas [X.Xiao etc., J.Virol., 70:8089-8108 (1996)].This discovery (hybridization signal that promptly has high-molecular-weight DNA) is the same with the PCR data, is to conform to integration behavior that when transduction takes place.RAAV integrated state described in this section and the I section is equally to genetically modified stability with prolong to express contribution is arranged.

Embodiment 7. gives Skeletal Muscle Cell rAAV and expresses ApoE

The following example has been described by rAAV carrier of the present invention is introduced and has been prolonged the expression of expressing another kind of therapeutic transgene product (apo E (ApoE), a kind of be used for the treatment of atherosclerotic protein) in the skeletal muscle.In addition, the prolongation that has allowed this transgene product that do not exist that destructive CTL replys is expressed.

The structure of A.rAAV

Make up the reorganization AAV carrier of coding secreted protein people ApoE according to the similar fashion of above-mentioned F. IX.Digest from plasmid pAlterApoE3 (Dr.Rader ' s Laboratory by the Xba I, University ofPennsylvania provides) go up to downcut ApoE cDNA, be cut into tack and be cloned in the pCMVLacZ skeleton of Not I digestion (seeing the vector construction block diagram of Figure 13).Separate the 2062bp Sma I/Sac I fragment that obtains lacZ gene among the pCMVLacZ, and the Sal I site of inserting this novel plasmid as stuffer.Separate acquisition ApoE minigene box (length overall 4.3kb) by EcoR I/Hind III digestion, this minigene box contains CMV promotor, ApoE cDNA SV40 polyadenylation sequence and 2062bp stuffer now, then this minigene box is connected in the pSub201 skeleton of Xba I digestion.Final product called after pSubCMVApoE-2062RO, with its when producing rAAV.ApoE according to the described step of rAAV.F. IX above as the cis plasmid.

B. the ApoE that expresses of analyzed in vitro rAAV.ApoE

The 84-31 cell inoculation is gone in 6 orifice plates, with rAAV.ApoE (in containing 2 milliliters of Dulbeccos Modified Eagles Medium of the 2% foetal calf serum) cells infected of 2 μ l CsCl purifying.Cell was cultivated 48 hours for 37 ℃.From the hole, take out the equal portions supernatant then with Western engram analysis ApoE.The result shows in the 84-31 cell conditioned medium liquid that infects with AAV.ApoE the ApoE albumen that obviously can survey is arranged.

C.rAAV.ApoE is in the intravital expression of ApoE knock-out mice

With 2.5-5 * 10 ¹⁰Individual particulate rAAV.ApoE is injected into that (every mouse accepts 5 * 10 altogether in the tibialis anterior muscle and quadriceps muscle of thigh of ApoE knock-out mice both sides ¹⁰To 5 * 10 ¹¹Individual particle).After injection the 28th day and the 120th day, even the prolongation that all available immunofluorescence detects local ApoE when the anti-ApoE antibody of blood plasma exists is expressed.

D. conclusion

Originally experimental results show that and carrier rAAV-ApoE intramuscularly is gone into to cause the myofiber transduction in the mouse that ApoE knocks out and secreted a large amount of recombinant proteins in the recycle system.Transgenosis is in the expression that does not have can prolong in the presence of the destructive CTL immunne response in myofiber, even excretory albumen can cause humoral immunization.

The intravital transgene expression of embodiment 8-primate

Anaesthetize a macaque, its forearm is clamped, sterilization, and the skin on tibialis anterior muscle is made the otch of 0.5cm.Determine manadesma, with rAV.CMVLacZ (embodiment 1 is described) viral suspension (175 μ l, 10 ²Genome/ml) is injected into manadesma 5-7mm depths.After 14 days, take out muscle biopsy's sample, be chilled among the OCT, cut into slices and dye with X-gal.With Leica Z500MC image processing and analytical system (Nikon FXA microscope interfaces is arranged) quantitative analysis tissue slice.The result of X-gal histological chemistry shows have high-caliber beta-galactosidase enzymes to express in most of myofibers of injection site.224mm in injection zone ²20% fiber expression beta-galactosidase enzymes is arranged in the area.According to the result of above-mentioned ApoE and F. IX, do not having under the cellular toxicity immunne response, expectation can prolong expression.These data show that genetically modified expression of the present invention can be reproduced in animal (especially primate) body beyond the mouse.

Many changes and variation that the present invention did are included at above-mentioned specification sheets, estimate that they are apparent to those skilled in the art.Think that these variations and change that method of the present invention is done are included in the scope of this paper claims.

Sequence table (1) general information:

(ⅰ) applicant: Trustees of the University of Pennsylvania

Wilson.James?M.

Fisher.Krishna?J.

(ⅱ) denomination of invention: the method for recombinant adeno-associated virus-directed gene therapy

(ⅲ) sequence number: 4

(ⅳ) mailing address:

(A) addressee: Howson and Howson

(B) street: Spring House Corporate Cntr, PO Box 457

(C) city: Spring House

(D) state: Pennsylvania

(E) country: USA

(F) postcode: 19477

(ⅴ) computer-reader form:

(A) recording medium type: floppy disk

(B) computer: IBM PC compatible type

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, Version#1.30

(ⅵ) the application's data:

(A) application number: WO

(B) applying date:

(C) classification:

(ⅶ) application materials formerly:

(A) application number: US 08/708.188

(B) applying date: on September 6th, 1996

(ⅶ)PRIOR?APPLICATION?DATA:

(A) application number: US 08/729.061

(B) applying date: on October 10th, 1996

(ⅷ) lawyer/proxy's information:

(A) name: Kodroff.Cathy A

(B) registration number: 33.980

(C) reference/file number: GNVPN 019CIP2PCT

(ⅸ) communication information:

(A) phone: 215-540-9200

(B) information of fax: 215-540-5818 (2) SEQ ID NO:1:

(ⅰ) sequence signature:

(A) length: 10398 base pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological framework: the unknown

(ⅱ) molecule type: cDNA

(Ⅹ ⅰ) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCGCTA GCATCATCAA TAATATACCT TATTTTGGAT TGAAGCCAAT ATGATAATGA 60 GGGGGTGGAG TTTGTGACGT GGCGCGGGGC GTGGGAACGG GGCGGGTGAC GTAGTAGTGT 120 GGCGGAAGTG TGATGTTGCA AGTGTGGCGG AACACATGTA AGCGACGGAT GTGGCAAAAG 180 TGACGTTTTT GGTGTGCGCC GGTGTACACA GGAAGTGACA ATTTTCGCGC GGTTTTAGGC 240 GGATGTTGTA GTAAATTTGG GCGTAACCGA GTAAGATTTG GCCATTTTCG CGGGAAAACT 300 GAATAAGAGG AAGTGAAATC TGAATAATTT TGTGTTACTC ATAGCGCGTA ATATTTGTCT 360 AGGGAGATCT GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC 420 GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA 480 ACTCCATCAC TAGGGGTTCC TTGTAGTTAA TGATTAACCC GCCATGCTAC TTATCTACAA 540 TTCGAGCTTG CATGCCTGCA GGTCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA 600 CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT AGTAACGCCA 660 ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCA 720 GTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG 780 CCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC 840 TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT 900 GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT 960 TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG 1020 ACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA TAAGCAGAGC TCGTTTAGTG 1080 AACCGTCAGA TCGCCTGGAG ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG 1140 GACCGATCCA GCCTCCGGAC TCTAGAGGAT CCGGTACTCG AGGAACTGAA AAACCAGAAA 1200 GTTAACTGGT AAGTTTAGTC TTTTTGTCTT TTATTTCAGG TCCCGGATCC GGTGGTGGTG 1260 CAAATCAAAG AACTGCTCCT CAGTGGATGT TGCCTTTACT TCTAGGCCTG TACGGAAGTG 1320 TTACTTCTGC TCTAAAAGCT GCGGAATTGT ACCCGCGGCC GCAATTCCCG GGGATCGAAA 1380 GAGCCTGCTA AAGCAAAAAA GAAGTCACCA TGTCGTTTAC TTTGACCAAC AAGAACGTGA 1440 TTTTCGTTGC CGGTCTGGGA GGCATTGGTC TGGACACCAG CAAGGAGCTG CTCAAGCGCG 1500 ATCCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA CTTAATCGCC 1560 TTGCAGCACA TCCCCCTTTC GCCAGCTGGC GTAATAGCGA AGAGGCCCGC ACCGATCGCC 1620 CTTCCCAACA GTTGCGCAGC CTGAATGGCG AATGGCGCTT TGCCTGGTTT CCGGCACCAG 1680 AAGCGGTGCC GGAAAGCTGG CTGGAGTGCG ATCTTCCTGA GGCCGATACT GTCGTCGTCC 1740 CCTCAAACTG GCAGATGCAC GGTTACGATG CGCCCATCTA CACCAACGTA ACCTATCCCA 1800 TTACGGTCAA TCCGCCGTTT GTTCCCACGG AGAATCCGAC GGGTTGTTAC TCGCTCACAT 1860 TTAATGTTGA TGAAAGCTGG CTACAGGAAG GCCAGACGCG AATTATTTTT GATGGCGTTA 1920 ACTCGGCGTT TCATCTGTGG TGCAACGGGC GCTGGGTCGG TTACGGCCAG GACAGTCGTT 1980 TGCCGTCTGA ATTTGACCTG AGCGCATTTT TACGCGCCGG AGAAAACCGC CTCGCGGTGA 2040 TGGTGCTGCG TTGGAGTGAC GGCAGTTATC TGGAAGATCA GGATATGTGG CGGATGAGCG 2100 GCATTTTCCG TGACGTCTCG TTGCTGCATA AACCGACTAC ACAAATCAGC GATTTCCATG 2160 TTGCCACTCG CTTTAATGAT GATTTCAGCC GCGCTGTACT GGAGGCTGAA GTTCAGATGT 2220 GCGGCGAGTT GCGTGACTAC CTACGGGTAA CAGTTTCTTT ATGGCAGGGT GAAACGCAGG 2280 TCGCCAGCGG CACCGCGCCT TTCGGCGGTG AAATTATCGA TGAGCGTGGT GGTTATGCCG 2340 ATCGCGTCAC ACTACGTCTG AACGTCGAAA ACCCGAAACT GTGGAGCGCC GAAATCCCGA 2400 ATCTCTATCG TGCGGTGGTT GAACTGCACA CCGCCGACGG CACGCTGATT GAAGCAGAAG 2460 CCTGCGATGT CGGTTTCCGC GAGGTGCGGA TTGAAAATGG TCTGCTGCTG CTGAACGGCA 2520 AGCCGTTGCT GATTCGAGGC GTTAACCGTC ACGAGCATCA TCCTCTGCAT GGTCAGGTCA 2580 TGGATGAGCA GACGATGGTG CAGGATATCC TGCTGATGAA GCAGAACAAC TTTAACGCCG 2640 TGCGCTGTTC GCATTATCCG AACCATCCGC TGTGGTACAC GCTGTGCGAC CGCTACGGCC 2700 TGTATGTGGT GGATGAAGCC AATATTGAAA CCCACGGCAT GGTGCCAATG AATCGTCTGA 2760 CCGATGATCC GCGCTGGCTA CCGGCGATGA GCGAACGCGT AACGCGAATG GTGCAGCGCG 2820 ATCGTAATCA CCCGAGTGTG ATCATCTGGT CGCTGGGGAA TGAATCAGGC CACGGCGCTA 2880 ATCACGACGC GCTGTATCGC TGGATCAAAT CTGTCGATCC TTCCCGCCCG GTGCAGTATG 2940 AAGGCGGCGG AGCCGACACC ACGGCCACCG ATATTATTTG CCCGATGTAC GCGCGCGTGG 3000 ATGAAGACCA GCCCTTCCCG GCTGTGCCGA AATGGTCCAT CAAAAAATGG CTTTCGCTAC 3060 CTGGAGAGAC GCGCCCGCTG ATCCTTTGCG AATACGCCCA CGCGATGGGT AACAGTCTTG 3120 GCGGTTTCGC TAAATACTGG CAGGCGTTTC GTCAGTATCC CCGTTTACAG GGCGGCTTCG 3180 TCTGGGACTG GGTGGATCAG TCGCTGATTA AATATGATGA AAACGGCAAC CCGTGGTCGG 3240 CTTACGGCGG TGATTTTGGC GATACGCCGA ACGATCGCCA GTTCTGTATG AACGGTCTGG 3300 TCTTTGCCGA CCGCACGCCG CATCCAGCGC TGACGGAAGC AAAACACCAG CAGCAGTTTT 3360 TCCAGTTCCG TTTATCCGGG CAAACCATCG AAGTGACCAG CGAATACCTG TTCCGTCATA 3420 GCGATAACGA GCTCCTGCAC TGGATGGTGG CGCTGGATGG TAAGCCGCTG GCAAGCGGTG 3480 AAGTGCCTCT GGATGTCGCT CCACAAGGTA AACAGTTGAT TGAACTGCCT GAACTACCGC 3540 AGCCGGAGAG CGCCGGGCAA CTCTGGCTCA CAGTACGCGT AGTGCAACCG AACGCGACCG 3600 CATGGTCAGA AGCCGGGCAC ATCAGCGCCT GGCAGCAGTG GCGTCTGGCG GAAAACCTCA 3660 GTGTGACGCT CCCCGCCGCG TCCCACGCCA TCCCGCATCT GACCACCAGC GAAATGGATT 3720 TTTGCATCGA GCTGGGTAAT AAGCGTTGGC AATTTAACCG CCAGTCAGGC TTTCTTTCAC 3780 AGATGTGGAT TGGCGATAAA AAACAACTGC TGACGCCGCT GCGCGATCAG TTCACCCGTG 3840 CACCGCTGGA TAACGACATT GGCGTAAGTG AAGCGACCCG CATTGACCCT AACGCCTGGG 3900 TCGAACGCTG GAAGGCGGCG GGCCATTACC AGGCCGAAGC AGCGTTGTTG CAGTGCACGG 3960 CAGATACACT TGCTGATGCG GTGCTGATTA CGACCGCTCA CGCGTGGCAG CATCAGGGGA 4020 AAACCTTATT TATCAGCCGG AAAACCTACC GGATTGATGG TAGTGGTCAA ATGGCGATTA 4080 CCGTTGATGT TGAAGTGGCG AGCGATACAC CGCATCCGGC GCGGATTGGC CTGAACTGCC 4140 AGCTGGCGCA GGTAGCAGAG CGGGTAAACT GGCTCGGATT AGGGCCGCAA GAAAACTATC 4200 CCGACCGCCT TACTGCCGCC TGTTTTGACC GCTGGGATCT GCCATTGTCA GACATGTATA 4260 CCCCGTACGT CTTCCCGAGC GAAAACGGTC TGCGCTGCGG GACGCGCGAA TTGAATTATG 4320 GCCCACACCA GTGGCGCGGC GACTTCCAGT TCAACATCAG CCGCTACAGT CAACAGCAAC 4380 TGATGGAAAC CAGCCATCGC CATCTGCTGC ACGCGGAAGA AGGCACATGG CTGAATATCG 4440 ACGGTTTCCA TATGGGGATT GGTGGCGACG ACTCCTGGAG CCCGTCAGTA TCGGCGGAAT 4500 TACAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA TAATAATAAC 4560 CGGGCAGGCC ATGTCTGCCC GTATTTCGCG TAAGGAAATC CATTATGTAC TATTTAAAAA 4620 ACACAAACTT TTGGATGTTC GGTTTATTCT TTTTCTTTTA CTTTTTTATC ATGGGAGCCT 4680 ACTTCCCGTT TTTCCCGATT TGGCTACATG ACATCAACCA TATCAGCAAA AGTGATACGG 4740 GTATTATTTT TGCCGCTATT TCTCTGTTCT CGCTATTATT CCAACCGCTG TTTGGTCTGC 4800 TTTCTGACAA ACTCGGCCTC GACTCTAGGC GGCCGCGGGG ATCCAGACAT GATAAGATAC 4860 ATTGATGAGT TTGGACAAAC CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA 4920 ATTTGTGATG CTATTGCTTT ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC 4980 AACAATTGCA TTCATTTTAT GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTCGGAT 5040 CCTCTAGAGT CGAGTAGATA AGTAGCATGG CGGGTTAATC ATTAACTACA AGGAACCCCT 5100 AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG CCGGGCGACC 5160 AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA GTGAGCGAGC GAGCGCGCAG 5220 CAGATCTGGA AGGTGCTGAG GTACGATGAG ACCCGCACCA GGTGCAGACC CTGCGAGTGT 5280 GGCGGTAAAC ATATTAGGAA CCAGCCTGTG ATGCTGGATG TGACCGAGGA GCTGAGGCCC 5340 GATCACTTGG TGCTGGCCTG CACCCGCGCT GAGTTTGGCT CTAGCGATGA AGATACAGAT 5400 TGAGGTACTG AAATGTGTGG GCGTGGCTTA AGGGTGGGAA AGAATATATA AGGTGGGGGT 5460 CTTATGTAGT TTTGTATCTG TTTTGCAGCA GCCGCCGCCG CCATGAGCAC CAACTCGTTT 5520 GATGGAAGCA TTGTGAGCTC ATATTTGACA ACGCGCATGC CCCCATGGGC CGGGGTGCGT 5580 CAGAATGTGA TGGGCTCCAG CATTGATGGT CGCCCCGTCC TGCCCGCAAA CTCTACTACC 5640 TTGACCTACG AGACCGTGTC TGGAACGCCG TTGGAGACTG CAGCCTCCGC CGCCGCTTCA 5700 GCCGCTGCAG CCACCGCCCG CGGGATTGTG ACTGACTTTG CTTTCCTGAG CCCGCTTGCA 5760 AGCAGTGCAG CTTCCCGTTC ATCCGCCCGC GATGACAAGT TGACGGCTCT TTTGGCACAA 5820 TTGGATTCTT TGACCCGGGA ACTTAATGTC GTTTCTCAGC AGCTGTTGGA TCTGCGCCAG 5880 CAGGTTTCTG CCCTGAAGGC TTCCTCCCCT CCCAATGCGG TTTAAAACAT AAATAAAAAA 5940 CCAGACTCTG TTTGGATTTG GATCAAGCAA GTGTCTTGCT GTCTTTATTT AGGGGTTTTG 6000 CGCGCGCGGT AGGCCCGGGA CCAGCGGTCT CGGTCGTTGA GGGTCCTGTG TATTTTTTCC 6060 AGGACGTGGT AAAGGTGACT CTGGATGTTC AGATACATGG GCATAAGCCC GTCTCTGGGG 6120 TGGAGGTAGC ACCACTGCAG AGCTTCATGC TGCGGGGTGG TGTTGTAGAT GATCCAGTCG 6180 TAGCAGGAGC GCTGGGCGTG GTGCCTAAAA ATGTCTTTCA GTAGCAAGCT GATTGCCAGG 6240 GGCAGGCCCT TGGTGTAAGT GTTTACAAAG CGGTTAAGCT GGGATGGGTG CATACGTGGG 6300 GATATGAGAT GCATCTTGGA CTGTATTTTT AGGTTGGCTA TGTTCCCAGC CATATCCCTC 6360 CGGGGATTCA TGTTGTGCAG AACCACCAGC ACAGTGTATC CGGTGCACTT GGGAAATTTG 6420 TCATGTAGCT TAGAAGGAAA TGCGTGGAAG AACTTGGAGA CGCCCTTGTG ACCTCCAAGA 6480 TTTTCCATGC ATTCGTCCAT AATGATGGCA ATGGGCCCAC GGGCGGCGGC CTGGGCGAAG 6540 ATATTTCTGG GATCACTAAC GTCATAGTTG TGTTCCAGGA TGAGATCGTC ATAGGCCATT 6600 TTTACAAAGC GCGGGCGGAG GGTGCCAGAC TGCGGTATAA TGGTTCCATC CGGCCCAGGG 6660 GCGTAGTTAC CCTCACAGAT TTGCATTTCC CACGCTTTGA GTTCAGATGG GGGGATCATG 6720 TCTACCTGCG GGGCGATGAA GAAAACGGTT TCCGGGGTAG GGGAGATCAG CTGGGAAGAA 6780 AGCAGGTTCC TGAGCAGCTG CGACTTACCG CAGCCGGTGG GCCCGTAAAT CACACCTATT 6840 ACCGGGTGCA ACTGGTAGTT AAGAGAGCTG CAGCTGCCGT CATCCCTGAG CAGGGGGGCC 6900 ACTTCGTTAA GCATGTCCCT GACTCGCATG TTTTCCCTGA CCAAATCCGC CAGAAGGCGC 6960 TCGCCGCCCA GCGATAGCAG TTCTTGCAAG GAAGCAAAGT TTTTCAACGG TTTGAGACCG 7020 TCCGCCGTAG GCATGCTTTT GAGCGTTTGA CCAAGCAGTT CCAGGCGGTC CCACAGCTCG 7080 GTCACCTGCT CTACGGCATC TCGATCCAGC ATATCTCCTC GTTTCGCGGG TTGGGGCGGC 7140 TTTCGCTGTA CGGCAGTAGT CGGTGCTCGT CCAGACGGGC CAGGGTCATG TCTTTCCACG 7200 GGCGCAGGGT CCTCGTCAGC GTAGTCTGGG TCACGGTGAA GGGGTGCGCT CCGGGCTGCG 7260 CGCTGGCCAG GGTGCGCTTG AGGCTGGTCC TGCTGGTGCT GAAGCGCTGC CGGTCTTCGC 7320 CCTGCGCGTC GGCCAGGTAG CATTTGACCA TGGTGTCATA GTCCAGCCCC TCCGCGGCGT 7380 GGCCCTTGGC GCGCAGCTTG CCCTTGGAGG AGGCGCCGCA CGAGGGGCAG TGCAGACTTT 7440 TGAGGGCGTA GAGCTTGGGC GCGAGAAATA CCGATTCCGG GGAGTAGGCA TCCGCGCCGC 7500 AGGCCCCGCA GACGGTCTCG CATTCCACGA GCCAGGTGAG CTCTGGCCGT TCGGGGTCAA 7560 AAACCAGGTT TCCCCCATGC TTTTTGATGC GTTTCTTACC TCTGGTTTCC ATGAGCCGGT 7620 GTCCACGCTC GGTGACGAAA AGGCTGTCCG TGTCCCCGTA TACAGACTTG AGAGGCCTGT 7680 CCTCGACCGA TGCCCTTGAG AGCCTTCAAC CCAGTCAGCT CCTTCCGGTG GGCGCGGGGC 7740 ATGACTATCG TCGCCGCACT TATGACTGTC TTCTTTATCA TGCAACTCGT AGGACAGGTG 7800 CCGGCAGCGC TCTGGGTCAT TTTCGGCGAG GACCGCTTTC GCTGGAGCGC GACGATGATC 7860 GGCCTGTCGC TTGCGGTATT CGGAATCTTG CACGCCCTCG CTCAAGCCTT CGTCACTGGT 7920 CCCGCCACCA AACGTTTCGG CGAGAAGCAG GCCATTATCG CCGGCATGGC GGCCGACGCG 7980 CTGGGCTACG TCTTGCTGGC GTTCGCGACG CGAGGCTGGA TGGCCTTCCC CATTATGATT 8040 CTTCTCGCTT CCGGCGGCAT CGGGATGCCC GCGTTGCAGG CCATGCTGTC CAGGCAGGTA 8100 GATGACGACC ATCAGGGACA GCTTCAAGGA TCGCTCGCGG CTCTTACCAG CCTAACTTCG 8160 ATCACTGGAC CGCTGATCGT CACGGCGATT TATGCCGCCT CGGCGAGCAC ATGGAACGGG 8220 TTGGCATGGA TTGTAGGCGC CGCCCTATAC CTTGTCTGCC TCCCCGCGTT GCGTCGCGGT 8280 GCATGGAGCC GGGCCACCTC GACCTGAATG GAAGCCGGCG GCACCTCGCT AACGGATTCA 8340 CCACTCCAAG AATTGGAGCC AATCAATTCT TGCGGAGAAC TGTGAATGCG CAAACCAACC 8400 CTTGGCAGAA CATATCCATC GCGTCCGCCA TCTCCAGCAG CCGCACGCGG CGCATCTCGG 8460 GCAGCGTTGG GTCCTGGCCA CGGGTGCGCA TGATCGTGCT CCTGTCGTTG AGGACCCGGC 8520 TAGGCTGGCG GGGTTGCCTT ACTGGTTAGC AGAATGAATC ACCGATACGC GAGCGAACGT 8580 GAAGCGACTG CTGCTGCAAA ACGTCTGCGA CCTGAGCAAC AACATGAATG GTCTTCGGTT 8640 TCCGTGTTTC GTAAAGTCTG GAAACGCGGA AGTCAGCGCC CTGCACCATT ATGTTCCGGA 8700 TCTGCATCGC AGGATGCTGC TGGCTACCCT GTGGAACACC TACATCTGTA TTAACGAAGC 8760 CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG 8820 GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT 8880 CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG 8940 ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC 9000 GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA 9060 AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT 9120 GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT 9180 TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA 9240 TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 9300 TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT 9360 ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA 9420 ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA 9480 CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA 9540 AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA 9600 GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTGC AGGCATCGTG 9660 GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA 9720 GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT 9780 GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT 9840 CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA 9900 TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAC ACGGGATAAT 9960 ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA 10020 AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC 10080 AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG 10140 CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC 10200 CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT 10260 GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA 10320 CCTGACGTCT AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG 10380 AGGCCCTTTC GTCTTCAA 10398 (2) SEQ ID NO: 2 of the message: ...

(ⅰ) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: the unknown

(ⅱ) molecule type: other nucleic acid

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:2:CATGGTAATA GCGATGACTA 20 (2) SEQ ID NO:3:

(ⅰ) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: the unknown

(ⅱ) molecule type: other nucleic acid

(ⅹ ⅰ) sequence description: the information of SEQ ID NO:3:GCTCTGCTTA TATAGACCTC 20 (2) SEQ ID NO:4:

(ⅰ) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: the unknown

(ⅱ) molecule type: other nucleic acid

(ⅹ ⅰ) sequence description: SEQ ID NO:4ATAAGCTGCA ATAAACAAGT 20

Claims (10)

1. recombinant adeno-associated virus rAAV is used for reducing application to the medicine of rAAV immunne response in production, this recombinant adeno-associated virus comprises heterologous gene, this heterologous gene is connected with its series of operations at cell inner expression of control, wherein rAAV is not subjected to the pollution of helper virus basically, and gives Skeletal Muscle Cell with it.

2. recombinant adeno-associated virus rAAV is used for prolonging the application of the medicine of transgene expression in production, this recombinant adeno-associated virus comprises transgenosis, this transgenosis is connected with its series of operations at cell inner expression of control, wherein rAAV is not subjected to the pollution of helper virus basically, and gives Skeletal Muscle Cell with it.

3. application according to claim 1 and 2, but wherein transgenosis is an excretory albumen.

4. application according to claim 3, wherein albumen is selected from factor IX, ApoE, beta-interferon, Regular Insulin, erythropoietin, tethelin and Rat parathyroid hormone 1-34.

5. according to the arbitrary described application of claim 1 to 4, wherein rAAV from 5 ' to 3 ' form by the reverse terminal repeat ITR of 5 ' AAV, allogeneic promoter, transgenosis, polyadenylation sequence and 3 ' AAV ITR.

6. application according to claim 1 and 2, wherein transgenosis is a dystrophin gene.

One kind in the presence of the cytotoxic immune that does not have anti-cell is replied in Skeletal Muscle Cell the method for express transgenic, this method comprises in the recombinant adeno-associated virus rAAV transfered cell, described rAAV comprises a transgenosis, this transgenosis is connected with the series of operations of its expression of control, wherein rAAV is not subjected to the pollution of helper virus basically, and wherein transgenosis is expressed in cell.

8. method according to claim 7, but wherein transgenosis is an excretory albumen.

9. method according to claim 8, wherein albumen is selected from factor IX, ApoE, beta-interferon, Regular Insulin, erythropoietin, tethelin and Rat parathyroid hormone 1-34.

10. method according to claim 7, wherein rAAV from 5 ' to 3 ' form by the reverse terminal repeat ITR of 5 ' AAV, allogeneic promoter, transgenosis, polyadenylation sequence and 3 ' AAV ITR.

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